Promoters of neural regeneration

ABSTRACT

The invention provides methods and compositions for promoting neural cell growth and/or regeneration. The general methods involve contacting with an activator of a cyclic nucleotide dependent protein kinase a neural cell subject to growth repulsion mediated by a neural cell growth repulsion factor. The activator may comprise a direct or an indirect activator of the protein kinase; the repulsion factor typically comprises one or more natural, endogenous proteins mediating localized repulsion or inhibition of neural cell growth; and the target cells are generally vertebrate neurons, typically injured mammalian neurons. The subject compositions include mixtures comprising a neural cell, an activator of a cyclic nucleotide dependent protein kinase and a neural cell growth repulsion factor.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35USC120 to U.S. Ser. No.09/900,268, filed on Jul. 6, 2001, which claims priority to U.S. Ser.No. 09/145,820, filed Sep. 2, 1998, now U.S. Pat. No. 6,268,352, whichare incorporated by reference herein in their entirety.

[0002] The research carried out in the subject application was supportedin part by NIH grant NS22764. The government may have rights in anypatent issuing on this application.

INTRODUCTION

[0003] 1. Field of the Invention

[0004] The field of this invention is nerve cell growth regulation.

[0005] 2. Background

[0006] The failure of the adult central nervous system (CNS) toregenerate after injury is a major clinical problem, affecting some200,000 people in the United States alone. Despite intensive research,an effective approach in promoting significant regeneration of CNS nervefibers remains lacking. The inability of CNS to regenerate is partly dueto inhibitory factors associated with myclin, a cellular structuresurrounding the nerve fibers.

[0007] There are currently few effective methods that can promotesignificant nerve regeneration of severed or damaged nerve fibers. Anumber of endogenous molecules are known to modulate neural cell growth(1). These factors may exert either attractive or repulsive action onthe extension of axonal growth cones (1,2). Experiments in mammals haveshown that blocking of some of the inhibitory factors by antibodiescould promote regeneration of severed axons in the spinal cord and leadto functional recovery of limb movements.

[0008] Recent reports have shown that attractive responses to netrins,mediated by the DCC/UNC-40 family of proteins, can be converted torepulsion by coexpression of proteins of the UNC-5 family (3). Inaddition, attractive effects of brain-derived neurotrophic factor (BDNF)and netrin-1 on Xenopus spinal neurites in culture can be converted torepulsion by inhibition of protein kinase A activity (4, 5). We disclosehere the opposite phenomenon: that by specific pharmacologicalmanipulations, the action of neural cell inhibitory (or repulsive)factors can be reversed. Such pharmacological treatments change theinhibitory nature of those inhibitory factors into supportive factorsand thus promote nerve regeneration.

SUMMARY OF THE INVENTION

[0009] We have found that the action of many protein factors that eitherinhibit or promote nerve growth are mediated through two generalmechanisms. Factors such as neurotrophins including brain-derivedneurotrophic factor (BDNF) and neurotrophin 3 (NT-3) as well as certainnetrins can promote nerve-growth in vitro. Nerve fibers grow towardsthese factors when presented as a localized source. On the other hand,factors such as Semaphorin III, myelin-associated glycoprotein (MAG) andpurified myelin, cause collapse of the nerve terminal and block nervegrowth. When these factors present as a localized source, nerve fibersgrow away from these factors. We have further found that the inhibitoryor repulsive effects of the latter factors can be reversed bypharmacological treatments that activate cyclic nucleotide dependentprotein kinases, e.g. by increasing the level of cyclic nucleotides,cAMP and cGMP. In addition, we found the action of protein factors thatpromote nerve growth, e.g. BDNF and NT-3, can also be enhanced by thesecyclic nucleotides. Thus by pharmacological interventions that elevatethe cyclic nucleotide levels, we are able to help nerve regenerationassociated with injuries of the nervous system.

[0010] Accordingly, the invention provides methods and compositions forpromoting neural cell growth and/or regeneration. The general methodsinvolve contacting with an activator of a cyclic nucleotide dependentprotein kinase a neural cell subject to growth repulsion mediated by aneural cell growth repulsion factor. The activator may comprise a director indirect activator of the protein kinase, including cyclic nucleotideanalogs, activators of a cyclic nucleotide cyclase, NO inducers,inhibitors of a cyclic nucleotide phosphodiesterase, etc. The repulsionfactor typically comprises one or more natural, endogenous proteinsmediating localized repulsion or inhibition of neural cell growth.Examples include neural cell guidance proteins such as semaphorins, CNSmyelin fractions or components thereof such as MAG, etc. The targetcells are generally vertebrate neurons, typically injured mammalianneurons in situ. The subject compositions include mixtures comprising aneural cell, an activator of a cyclic nucleotide dependent proteinkinase and a neural cell growth repulsion factor.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIGS. 1A, 1B. (A) The dependence of turning responses on the SemaIII concentration in the pipette. Turning angles (mean±SEM) (8) innormal medium (filled symbols) and in medium containing 8-br-cGMP (100μM, open symbols), and for heat-inactivated Sema III (squares). Thetotal number of neurons examined is shown in parenthesis. ‘*’:Significantly different from the data set at zero concentration (p<0.05,Kruskal-Wallis test). (B) Correlation of turning angles induced by aSema III gradient in normal medium (first turn) and in medium containing8-br-cGMP (second turn) for the same neuron. The line represents bestlinear fit of the data (r=−0.88, p=0.02).

[0012] FIGS. 2A-2D. Growth cone turning in a gradient of Sema III. (A)Effects of manipulating cGMP-dependent activities. Angular positions ofall growth cones at the end of the one-hour exposure to a Sema IIIgradient (50 μg/ml in the pipette) are shown in a cumulativedistribution plot for the following conditions: normal medium, mediumcontaining 8-br-cGMP (100 μM), Sp-cGMPS (10 μM), Protoporphyrin-9 (PP-9,10 μM), S-Nitroso-N-acetylpenicillamine (SNAP, 300 μM), and Rp-cGMPS (10μM). Isolated symbols along the abscissa are median values forcorresponding data shown above.(B) Effects of manipulatingcAMP-dependent activities. Shown are distributions of turning angles forthe following conditions: normal culture medium, medium containing8-br-cGMP (100 μM), Rp-cAMPS (20 μM), Sp-cAMPS (20 μM), both 8-br-cGMP(100 μM) and Rp-cAMPS (20 μM), or both 8-br-cGMP (100 μM) and Sp-cAMPS(20 μM). (C) Distribution of turning angles in the absence or presenceof α-28 (20 μg/ml) in normal medium and in medium containing 8-br-cGMP(100 μM). ‘*’: significantly different (p<0.01, Kolmogorov-Smirnovtest). (D) Effects of reducing [Ca²⁺]_(o) on turning responses in a SemaIII gradient. Due to increased growth rate, the turning in 1 μM[Ca²⁺]_(o) was assayed 30 min after the onset of the gradient.Distribution of turning angles in normal (1 mM) or low (1 μM) Ca²⁺medium in the absence or presence of 8-br-cGMP (100 μM). ‘‡’: notsignificantly different (p>0.2, Kolmogorov-Smirnov test).

[0013]FIG. 3. Percentage of intact growth cones from the explantstreated with cyclic nucleotides and/or Sema III. Error bars refer toSEM. ‘*’: Significantly different from the set without pretreatment with8-br-cGMP and Sp-cAMPS (P<0.001, t-test)

[0014] FIGS. 4A-4D. (A) Effects of manipulating cyclic nucleotide levelson turning induced by a rMAG gradient (150 μg/ml in the pipette) innormal medium, in medium containing 8-br-cGMP (100 μM) or Sp-cAMPS (20μM), and by heat-inactivated rMAG in normal medium. Results from thelatter were significantly different from all three other groups (p<0.01,Kolmogorov-Smirnov test). (B) Effects of reducing [Ca²⁺]_(o) on turningresponses induced by rMAG. Distribution of turning angles in normal (1mM) or low (1 μM) Ca²⁺ medium in the absence or presence of Sp-cAMPS (20μM). ‘*’: Significantly different (p<0.05, Kolmogorov-Smirnov test). (C)Turning responses induced by a gradient of netrin-1 (5 μg/ml in thepipette) (5), in normal medium and in medium containing Rp-cGMPS (10EM), 8-br-cGMP (100 μM), Rp-cAMPS (20 μM), or both Rp-cAMPS (20 μM) and8-br-cGMP (100 μM). Also shown is the control distribution of turningangles observed when the pipette contained only culture medium (nogradient), which is significantly different from all other data sets(p<0.05, Kolmogorov-Smirnov test). (D) Turning induced by a NT-3gradient (50 μg/ml in the pipette) in normal medium and in mediumcontaining Rp-cAMPS (20 μM) or Rp-cGMPS (10 μM), or in medium containing1 μM Ca²⁺. All data sets were significantly different from the nogradient control (p<0.05, Kolmogorov-Smirnov test).

[0015]FIG. 5. Growth cone turning induced by purified myelin. A gradientof purified myelin (25 μg/ml in the pipette) was applied in normalculture medium (open circles), in medium containing Sp-cAMPS (20 μM,solid circles), or 8-br-cGMP (100 μM, solid squares). Isolated symbolsalong the abscissa are median values for corresponding data shown above.

[0016]FIGS. 6A, 6B. Effects of purified myelin-associated glycoprotein(MAG) on growth cones of Xenopus spinal neurons. A gradient of purifiedMAG (150 μg/ml in the pipette) was applied in normal culture medium(open circles), in medium containing Sp-cAMPS (20 μM, solid circles), orin medium containing 8-br-cGMP (100 μM, solid squares). (A) Shown aredistributions of turning angles under different conditions. Isolatedsymbols along the abscissa are median values for corresponding datashown above. (B) Shown are distributions of net neurite extension in thegradient under different conditions.

[0017]FIGS. 7A, 7B. Effects of reducing external Ca²⁺. (A) PurifiedMAG-induced turning depends on [Ca^(2+]) _(o). Shown are distributionsof turning angles in normal medium in the absence (solid circles) orpresence of Sp-cAMPS (20 μM, solid squares), and in medium containing 1μM [Ca²⁺]_(o), in the absence (open circles) or presence of Sp-cAMPS (20μM, open squares), respectively. Isolated symbols along the abscissa aremedian values for corresponding data shown above. (B) Purifiedmyelin-induced turning depends on [Ca²⁺]_(o). Shown are distributions ofturning angles in normal medium in the absence (solid circles) orpresence of Sp-cAMPS (20 μM, solid squares), and in medium containing 1μM [Ca²⁺]_(o) in the absence (open circles) or presence of Sp-cAMPS (20μM, open squares), respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The general methods involve contacting with an activator of acyclic nucleotide dependent protein kinase a neural cell subject togrowth repulsion mediated by a neural cell growth repulsion factor.Preferred activators enhance the activity of at least one of PKA or PKG.A wide variety of direct and indirect activators of cyclic nucleotidedependent protein kinases are known in the art, or readily identified inassays such as immuno, kinase and cell based assays. Indirect activatorsare agents which increase the activity of the protein kinase withoutdirectly interacting with the kinase, and include any agent whichincreases the functional activity of the corresponding cyclic nucleotide(e.g. by increasing its synthesis, increasing its availability,decreasing its degradation, etc.). Exemplary activators include cyclicnucleotide analog agonists, activators of cyclic nucleotide cyclases, NOinducers, inhibitors of cyclic nucleotide phosphodiesterases, drugs suchas KT5720, etc. Additional activators are readily made by screeningcandidate agents for activation of the targeted protein kinase,inhibition of a targeted phosphodiesterase (e.g. cAMP or cGMPphosphodiesterase), activation of a targeted cyclase (e.g. guanylate oradenylate cyclase), etc. in conventional in vitro or cell based assays.

[0019] The repulsion factor typically comprises one or more natural,endogenous agents mediating localized repulsion or inhibition of thetargeted neural cell growth, which repulsion or inhibition is reversibleby increasing the activity of a cyclic nucleotide dependent proteinkinase in the cell. Such factors are generally present at the site ofneuronal cells in situ, particularly at the cite of CNS axons, andprovide an endogenous inhibition to nerve cell growth and/orregeneration. A wide variety of such factors are known or are readilyidentified in cell based assays, such as described herein. Exemplaryagents capable of acting as repulsion factors include neural cellguidance proteins such as some semaphorins, netrins, CNS myclinfractions or components thereof such as MAG, etc.

[0020] The target cells are generally vertebrate neurons, typicallyinjured mammalian neurons in situ. A wide variety of methods may be usedto effect the contacting of the cell with the activator. For example,for CNS administration, a variety of techniques are available forpromoting transfer of therapeutic agents across the blood brain barrierincluding disruption by surgery or injection, drugs which transientlyopen adhesion contact between CNS vasculature endothelial cells, andcompounds which facilitate translocation through such cells. Thecompositions may also be amenable to direct injection or infusion,intraocular administration, or within/on implants e.g. fibers such ascollagen fibers, in osmotic pumps, grafts comprising appropriatelytransformed cells, etc.

[0021] In a preferred embodiment, the activator is delivered locally andits distribution is restricted. For example, a particular method ofadministration involves coating, embedding or derivatizing fibers, suchas collagen fibers, protein polymers, etc. with therapeutic agents, seealso Otto et al. (1989) J Neuroscience Research 22, 83-91 and Otto andUnsicker (1990) J Neuroscience 10, 1912-1921. Another particularembodiment is adapted from treatment of spinal cord injuries, e.g.Schulz M K, et al., Exp Neurol. 1998 February; 149(2): 390-397; Guest JD, et al., J Neurosci Res. Dec. 1, 1997; 50(5): 888-905; Schwab M E, etal., Spinal Cord. 1997 July; 35(7): 469-473; Tatagiba M, et al.,Neurosurgery. 1997 March; 40(3): 541-546. For example, the subjectcompositions improve corticospinal tract (CST) regeneration followingthoracic spinal cord injury by promoting CST regeneration into humanSchwann cell grafts in the methods of Guest et al. (supra). For thesedata, the human grafts are placed to span a midthoracic spinal cordtransection in the adult nude rat, a xenograft tolerant strain.Activators (see Table 1) incorporated into a fibrin glue are placed inthe same region. Anterograde tracing from the motor cortex using thedextran amine tracers, Fluororuby (FR) and biotinylated dextran amine(BDA), are performed. Thirty-five days after grafting, the CST responseis evaluated qualitatively by looking for regenerated CST fibers in orbeyond grafts and quantitatively by constructing camera lucidacomposites to determine the sprouting index (SI), the position of themaximum termination density (MTD) rostral to the GFAP-defined host/graftinterface, and the longitudinal spread (LS) of bulbous end terminals.The latter two measures provide information about axonal die-back. Incontrol animals (graft only), the CST do not enter the SC graft andundergo axonal die-back. As shown in Table 1, the activatorsdramatically reduce axonal die-back and cause sprouting. TABLE I In VivoNeuronal Regeneration with Exemplary Activator Formulations ReducedPromote Activator Formulation Die-Back Sprouting  1. Forskolin 5 uM ++++++++  2. 7β-Deaceyl-7β-[γ- 5 uM ++++ ++++ (morpholino)butyryl]-forskolin 3. 6β-[β′-(Piperidino)- 5 uM ++++ ++++ propionyl]-forskolin  4.3-Isobutyl-1-methyl- 25-100 uM ++++ ++++ xanthine(IBMX)  5. Rolipram 2uM ++++ ++++  6. 8-bromo-cAMP 100 uM ++++ ++++  7. 8-chloro-cAMP 100 uM++++ ++++  8. 8-(4-chlorophenylthio)-cAMP 100 uM ++++ ++++  9.Dibutyryl-cAMP 100 uM ++++ ++++ 10. Dioctanoyl-cAMP 100 uM ++++ ++++ 11.Sp-cAMPS 20 uM ++++ ++++ 12. Sp-8-bromo-cAMPS 20 uM ++++ ++++ 13.8-br-cGMP 100 uM ++++ ++++ 14. 8-(4-chlorophenylthio)-cGMP 100 uM ++++++++ 15. Dibutyryl-cGMP 100 uM ++++ ++++ 16. Glyco-SNAP-1 300 uM ++++++++ 17. Glyco-SNAP-2 300 uM ++++ ++++ 18. S-Nitroso-N-acetylpenicill-300 uM ++++ ++++ amine 19. NOC-18 100 uM ++++ ++++ 20. NOR-3 100 uM ++++++++ 21. Protoporphyrin-9 10 uM ++++ ++++

[0022] In another demonstration of in vivo therapeutic activity, thesubject activators are incorporated in the implantable devices describedin U.S. Pat No. 5,656,605 and tested for the promotion of in vivoregeneration of peripheral nerves. Prior to surgery, 18 mmsurgical-grade silicon rubber tubes (I.D. 1.5 mm) are prepared with orwithout guiding filaments (four 10-0 monofilament nylon) and filled withtest compositions comprising the activators of Table 1. Experimentalgroups consist of: 1. Guiding tubes plus Biomatrix 1™ (BiomedicalTechnologies, Inc., Stoughton, Mass.); 2. Guiding tubes plus Biomatrixplus filaments; 3-23. Guiding tubes plus Biomatrix 1™ plus activators1-21 of Table 1 (supra).

[0023] The sciatic nerves of rats are sharply transected at mid-thighand guide tubes containing the test substances with and without guidingfilaments sutured over distances of approximately 2 mm to the end of thenerves. In each experiment, the other end of the guide tube is leftopen. This model simulates a severe nerve injury in which no contactwith the distal end of the nerve is present.

[0024] After four weeks, the distance of regeneration of axons withinthe guide tube is tested in the surviving animals using a functionalpinch test. In this test, the guide tube is pinched with fine forceps tomechanically stimulate sensory axons. Testing is initiated at the distalend of the guide tube and advanced proximally until muscularcontractions are noted in the lightly anesthetized animal. The distancefrom the proximal nerve transection point is the parameter measured. Forhistological analysis, the guide tube containing the regenerated nerveis preserved with a fixative. Cross sections are prepared at a pointapproximately 7 mm from the transection site. The diameter of theregenerated nerve and the number of myclinated axons observable at thispoint are used as parameters for comparison.

[0025] Measurements of the distance of nerve regeneration document thetherapeutic effect of groups 3-23. Similarly, plots of the diameter ofthe regenerated nerve measured at a distance of 7 mm into the guide tubeas a function of the presence or absence of one or more activators ofthe device demonstrate a similar therapeutic effect of all 21 activatorstested. No detectable nerve growth is measured at the point sampled inthe guide tube with the matrix-forming material alone. The presence ofguiding filaments plus the matrix-forming material (no activator)induces only very minimal regeneration at the 7 mm measurement point,whereas dramatic results, as assessed by the diameter of theregenerating nerve, are produced by the device which consisted of theguide tube, guiding filaments and activator compositions. Finally,treatments using guide tubes comprising either a matrix-forming materialalone, or a matrix-forming material in the presence of guidingfilaments, result in no measured growth of myelinated axons. Incontrast, treatments using a device comprising guide tubes, guidingfilaments, and matrix containing activator compositions consistentlyresult in axon regeneration, with the measured number of axons beingincreased markedly by the presence of guiding filaments.

[0026] The amount of activator administered depends on the activator,formulation, route of administration, etc. and is generally empiricallydetermined. For example, with cyclic nucleotide activators deliveredlocally in a solid matrix or semi-solid phase, the administered dose istypically in the range of about 2 mg up to about 2,000 mg, althoughvariations will necessarily occur depending on the target, the host, andthe route of administration, etc.

[0027] In one embodiment, the invention provides the subject activatorscombined with a pharmaceutically acceptable excipient suitable forcontacting target neuronal cells in situ, such as CNS administration,including as sterile saline or other medium, gelatin, an oil, etc. toform pharmaceutically acceptable compositions. The compositions and/orcompounds may be administered alone or in combination with anyconvenient carrier, solid or semi-solid matrix, diluent, etc. and suchadministration may be provided in single or multiple dosages. Usefulcarriers and matrices include solid, semi-solid or liquid mediaincluding water and non-toxic organic solvents. In another embodiment,the invention provides the subject compounds in the form of a pro-drug,which can be metabolically converted to the subject compound by therecipient host. A wide variety of pro-drug formulations are known in theart. The compositions may be provided in any convenient form includingtablets, capsules, fibers, guides, osmotic pumps, etc. (see, e.g. U.S.Pat. Nos. 5,656,605; 5,660,849 and 5,735,863 for delivery systemsparticularly suited for CNS administration). As such the compositions,in pharmaceutically acceptable dosage units or in bulk, may beincorporated into a wide variety of containers and materials. Forexample, dosage units may be included in a variety of containersincluding microcapsules, pumps, fibers, etc.

[0028] The compositions may be advantageously combined and/or used incombination with other therapeutic or prophylactic agents, differentfrom the subject compounds. In many instances, administration inconjunction with the subject compositions enhances the efficacy of suchagents. For example, the compounds may be advantageously used inconjunction with other neurogenic agents, neurotrophic factors, growthfactors, anti-inflammatories, antibiotics etc.; and mixtures thereof,see e.g. Goodman & Gilman 's The Pharmacological Basis of Therapeutics,9^(th) Ed., 1996, McGraw-Hill, esp. Chabner et al., AntineoplasticAgents at pp.1233.

[0029] The subject compositions include ex vivo mixtures comprising aneural cell, an activator of a cyclic nucleotide dependent proteinkinase and a neural cell growth repulsion factor. Such mixtures may beused in in vitro screens for identifying suitable activators, optimizingformulations, delivery concentrations, etc., etc.

[0030] The following experimental section and examples are offered byway of illustration and not by way of limitation.

EXAMPLES

[0031] Collapsin-1/Semaphorin III/D (Sema III), a diffusible member ofthe semaphorin family can repel or cause collapse of growth cones inculture (6). Defects in Sema III knock-out mice suggest that Sema IIIcreates exclusion zones for axons or drives axonal fasciculation throughsurround repulsion (7). We analyzed the effect of a microscopic gradientof Sema III on growth cones of the cultured Xenopus spinal neuron (8).Sema III-containing saline was applied in pulses from a micropipettepositioned 100 μm from the center of the growth cone and at a 45° anglewith respect to the original direction of neurite extension. Most growthcones grew away from the pipette. The repulsive response wasdose-dependent with a minimal response occurring at an effectiveconcentration of about 10 ng/ml at the growth cone (8). Heat-inactivatedSema III was ineffective (FIG. 1A). The repulsive turning was initiatedby active protrusion of filopodia in the direction away from thepipette, with no obvious growth cone collapse during the turning process(9). The rate of neurite extension was unaffected by the presence of theSema III gradient.

[0032] When 8-br-cGMP (10) or Sp-cGMPS (11), membrane permeable agonistsof endogenous cGMP signaling pathways, was present in the culture medium(e.g. a gradient of Sema III −50 μg/ml in the pipette-applied in normalculture medium or in medium containing 100 μM 8-br-cGMP), nearly allgrowth cones turned toward rather than away from the pipette in the sameSema m gradient (FIG. 2A). Protoporphyrin-9 (PP-9), a guanylate cyclaseactivator (12), has similar effect (FIG. 2A). Application of a nitricoxide (NO) donor S-Nitroso-N-acetylpenicillamine (SNAP), which activatessoluble guanylate cyclase by releasing NO (13), abolished the repulsiveturning response without causing a significant attractive response. Onthe other hand, bath application of Rp-cGMPS (11), a cGMP antagonist anda specific inhibitor of protein kinase G, did not affect the growth coneresponse. Thus cGMP regulates the direction of growth cone turninginduced by Sema III. NO and cGMP can regulate the establishment of thecentral connections of developing retinal axons and stimulate thesynapse formation of developing and regenerating olfactory neurons (14).In contrast to the effect of cGMP analogues, we found that cAMPanalogues had no significant effect on the repulsion induced by Sema IIIgradients (FIG. 2B). However, a cAMP antagonist Rp-cAMPS, but notagonist Sp-cAMPS (15), blocked the conversion of the turning response inthe presence of 8-br-cGMP (FIG. 2B), indicating interaction betweencAMP- and cGMP-dependent pathways in these neurons.

[0033] The opposite turning responses are not due to behaviors ofdifferent types of neurons in these Xenopus cultures (16), and theturning response can be converted in the same neuron. After a repulsiveturning response was first elicited by a Sema III gradient, the sameneuron was tested by the same gradient in the presence of 8-br-cGMP (100μM). We found that the growth cone exhibited an attractive response.Repulsive response was restored after 8-br-cGMP was washed away. Thusthe response of the growth cone to a Sema III gradient does notdesensitize with time and can be switched between repulsion andattraction in a cGMP-dependent manner. The extent of repulsion versusattraction for each individual neuron appears to be correlated (FIG.1B), indicating that cGMP affects only the directionality of theresponse, not the extent to which the growth cone turns.

[0034] Neuropilins are receptors for several members of the semaphorinfamily (17-19), and antibodies against the extracellular domain ofneuropilin-1 blocks the effects of Sema III in vitro (17, 18). Afunction-blocking antiserum to rat neuropilin-1 (α-28) cross-reacts withthe Xenopus protein (20). We found that bath application of the cc-28antiserum abolished both the repulsion induced by Sema III under normalconditions and the attraction towards Sema III in the presence of8-br-cGMP (FIG. 2C). Thus neuropilin-1 function is required for bothrepulsion and attraction of these growth cones induced by Sema III.

[0035] Cyclic nucleotides also change the responsiveness of developingrat dorsal root ganglion (DRG) axons to Sema III. When added to culturesof DRG explants (21), 8-br-cGMP, but not Sp-cAMPS, inhibited thecollapsing activity of Sema III in a dose-dependent manner (FIG. 3),while 8-br-cGMP or Sp-cAMPS alone had no detectable effect on growthcones in these cultures. In these experiments higher concentrations of8-br-cGMP (0.5-5 mM) were needed to inhibit the collapsing activity ofSema III, suggesting that the turning response of Xenopus spinal neuronsmight be more sensitive to modulation by cGMP than is the collapse ofgrowth cones of rat DRG neurons (9).

[0036] To examine whether the responses to other repulsive factors canbe converted, we studied myclin-associated glycoprotein (MAG), acomponent of myclin and an inhibitor of axonal regeneration (22). Asoluble proteolytic fragment of MAG consisting of its extracellulardomain is released in abundance from myelin in vivo, and can potentlyinhibit axon regeneration (23). We found that a gradient of recombinantprotein consisting of the extracellular domain of MAG (rMAG) (24)repelled growth cones of Xenopus spinal neurons (FIG. 4A). However, therepulsion by rMAG was not affected by the addition of 8-br-cGMP. On theother hand, when the Sp-cAMPS was added to the medium, the growth coneresponses were converted to attraction in the same rMAG gradient (FIG.4A). Thus the turning response induced by rMAG can be modulated bycAMP-dependent activities. This is reminiscent of the turning responsesinduced by gradients of BDNF and netrin-1 (4, 5), although the latterfactors are normally attractive and are converted to repulsive byinhibition of cAMP-dependent activities.

[0037] Cytosolic Ca²⁺ regulates growth cone motility (25). An increasein cytosolic Ca²⁺ levels correlate with growth cone collapse induced bysome myelin-associated proteins, but not other factors (26). To examinethe involvement of Ca²⁺ in Sema III and rMAG-induced turning, we reducedthe extracellular Ca²⁺ concentration ([Ca²⁺]_(o)) from the normal levelof 1 mM to 1 μM (4). This resulted in a 2-3 fold increase in the rate ofneurite extension, but no change in the turning responses in a Sema IIIgradient (FIG. 2D). In contrast, the repulsion and attraction induced bygradients of rMAG were both abolished by the reduction of [Ca²⁺]_(o)(FIG. 4B). Thus growth cone turning induced by rMAG, but not Sema III,requires normal [Ca²⁺]_(o).

[0038] The above studies on growth cone turning induced by Sema m andMAG point to the existence of two distinct pathways involving cAMP andcGMP, with different Ca²⁺-dependences. Growth cone turning induced byBDNF, acetylcholine, and netrin-1, but not NT-3, depends on both[Ca²⁺]_(o) and cAMP (4, 5). Here, we found that inhibition or activationof cGMP-dependent pathways by Rp-cGMPS or 8-br-cGMP, respectively, didnot affect the attractive turning towards netrin-1 (p>0.1,Kolmogorov-Smirnov test) (FIG. 4C). Moreover, repulsive turning inducedby the same netrin-1 gradient in the presence of Rp-cAMPS was also notaffected by 8-br-cGMP. On the other hand, the attractive response in aNT-3 gradient was converted to repulsive response by inhibitingcGMP-dependence pathways with Rp-cGMPS, while depletion of [Ca²⁺]_(o) oraddition of Rp-cAMPS had no effect on the attractive turning (FIG. 4D).Thus, the dependence of turning behavior on [Ca²⁺]_(o) for the fourfactors examined here (rMAG, netrin-1, Sema III and NT-3) correlateswith a dependence on cAMP, not cGMP.

[0039] In addition to recombinant Collapsin I/Semaphorin III (Sema III)and myelin-associated glycoprotein (rMAG, consisting of theextracellular domain of MAG), we also tested purified myelin-associatedglycoprotein (MAG) and myelin (which have many components) and obtainedresults similar to those with rMAG (see, FIGS. 5-7). For these studies,meylin was made from bovine brain corpus callosum. The tissue was firsthomogenized. The homogenate then was filtered through a cheese cloth andoverlaid on 0.85M sucrose and centrifuged for 25 min at 75,000 g. Thematerial at the interface was collected and resuspended in ice-water andcentrifuged for 15 min at 10,000 g. This step was repeated once more,and the purified myelin was collected and washed twice with ice-water.To obtain the soluble form of myelin protein, the myelin fractions weresolubilized in 60 mM octylglucoside at 10 mg/ml at 4° C. for 1 hr. Thesupernatants were collected and dialyzed against PBS and F12 medium.Native MAG was purified from myelin after extraction in 1%octylglucoside and separation by DEAE-Sepharose column as described inMcKerracher et al., Neuron 13, 805-811, 1994 and Li et al., Journal ofNeuroscience Research, 46, 404-414, 1996.

[0040] Studies of the turning response of Xenopus neuronal growth conesinduced by a number of diffusible factors (4, 5), including thoseexamined here, have implicated cAMP and cGMP in setting the neuronalresponse to different guidance cues. The guidance cues examined can allbe either attractive or repulsive, depending on the status of cytosoliccyclic nucleotides. Manipulations to increase the level of cyclicnucleotide activity favor attraction and manipulations to decrease thelevel of cyclic nucleotide activity favor repulsion. Since cyclicnucleotides are known to serve as second messengers for a large numberof cell surface receptors (27), the response of a growth cone to aparticular guidance cue may thus depend critically on other coincidentsignals received by the neuron. The susceptibility to conversion betweenattraction and repulsion may enable a growing axon to responddifferentially to the same guidance cue at different points along itsjourney to its final target (28). The reversal of the action ofrepulsive factors by elevated cyclic nucleotides provides means forpromoting nerve regeneration in the central nervous system (CNS), sinceeffective regeneration in the CNS is blocked by inhibitory factors (22,29), and modulation of cyclic nucleotide levels helps relieve thisinhibition and therefore help stimulate regeneration.

PARENTHETICAL REFERENCES AND NOTES

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[0048] 8. Dissociated Xenopus spinal neurons were prepared as described(4,5). Isolated spinal neurons were used for experiments at roomtemperature (22°-24° C.) 14-22 hour after plating. Sema III was purifiedfrom conditioned medium of stable 293-EBNA cell lines secreting SemaIII-AP as described (17). To inactivate Sema III activity, thesupernatant containing Sema III-AP was heated at 85° C. for 45 minutes[J. Fan and J. A. Raper, Neuron 14, 263 (1995)]. Microscopic gradientsof diffusible factors were produced as described (4,5). The averageconcentration of factors at the growth cone was about 10³-fold lowerthan that in the pipette [A. M. Lohof, M. Quilian, Y. Dan and M-m. Poo,J. Neurosci. 12, 1253 (1992)], and a concentration gradient of 5-10% wascreated across a growth cone 10 μm wide, 100 μm away from the tip of theejecting pipette. The original direction of neurite growth was definedby the last 10-μm segment of the neurite. The turning angle was definedby the angle between the original direction of neurite extension and astraight line connecting the positions of the growth cone at the onsetand the end of the one hour period. Only growth cones with a netextension >5 μm over the one-hour period were scored.

[0049] 9. No obvious collapse of growth cones was observed even when ahigher concentration (up to 100 μg/ml of Sema III in the pipette) wasapplied to cultured Xenopus spinal neurons. We have not examined whethera uniform concentration of Sema III causes collapse, as it does forchick and rat growth cones.

[0050] 10. M. A. Schwarzschild and R. E. Zigmond, J. Neurochem. 56, 400(1991). Pharmacological agents were added to the culture medium at least30 minutes before the gradient was applied and were present during theexperiments. The final concentrations of pharmacological agents in themedium were listed.

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[0056] 16. These Xenopus cultures contain spinal neurons sensitive tovarious neurotransmitters [J. L. Bixby and N. C. Spitzer, J. Physiol.353, 143 (1984)] exhibiting a wide-range of capability in secretingacetylcholine [J. Evers, M. Laser, Y. A. Sun, Z. P. Xie, M-m. Poo, J.Neurosci. 9, 1523 (1989)]. The variability of turning responses observedwithin a population of these neurons (see FIG. 2) could be attributed toeither the heterogeneity of neuronal types or variability in thecytoplasmic level of cyclic nucleotides or other signal transductioncomponents.

[0057] 17. Z. He and M. Tessier-Lavigne, Cell 90, 739 (1997).

[0058] 18. A. L. Kolodkin et al., ibid. p. 753.

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[0060] 20. The anti-neuropilin-1 antibody (α-28) is directed against theectodomain of rat neuropilin-1, purified on a protein-A agarose columnas described (17), and does not cross-react with neuropilin-2 (19).Using α-28, a putative Xenopus neuropilin was detected by Western blot.Interaction of α-28 with putative Xenopus neuropilin in cultured Xenopusspinal neurons was confirmed by immunostaining. For antibody blockingexperiments, α-28 (20 μg/ml) were added 30 minutes before the onset ofthe gradient. During the turning assay, the concentration of α-28 was 5μg/ml.

[0061] 21. DRG explants derived from E14 rat embryos were cultured with25 ng/ml NGF on plates precoated with poly-D-lysine and laminin for 20hours before experiments as described (17). Pharmacological agents wereadded an hour before the collapse assay. The collapse assay wasperformed on sensory axons from these explants using SemaIII-AP-containing medium essentially as described (17). Forvisualization, growth cones were stained with rhodamine-phalloidin, thenwashed and mounted.

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[0064] 24. To generate rMAG, SF9 cells were infected with baculovirusexpressing the extracellular domain of MAG and conditioned medium wascollected 5-6 days after infection and purified as described [M. Li etal., J. Neurosci. Res. 46, 404 (1996)]. For experiments usinginactivated rMAG, rMAG was heated at 80° C. for 35 min.

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[0067] 27. J. R. Cooper, F. E. Bloom, R. H. Roth, The Biochemical Basisof Neuropharmacology (7th ed, Oxford University Press, New York, 1996);R. Laufer and J. Changeux, EMBO J. 6, 901 (1987); J. H. Fong and D. E.Ingber, Biochem. Biophys. Res. Commun. 221, 19 (1996).

[0068] 28. Attraction of commissural axons towards the floor plate inthe developing spinal cord might be switched off (or even converted to arepulsive response) after the contact with the floor plate cells,allowing further axonal growth past the floor plate [see R. Shirasaki,R. Katsumata, F. Murakami, Science 279, 105 (1998)].

[0069] 29. M. E. Schwab and D. Bartholdi, Physiol. Rev. 76, 319 (1996).

What is claimed is:
 1. A method for promoting growth of an adult humancentral nervous system neuron damaged by a spinal injury and subject togrowth inhibition by an endogenous neural cell growth repulsion factor,the method comprising the steps of locally administering to an adulthuman patient in need thereof at an axon of the neuron a therapeuticallyeffective amount of an activator of a cyclic nucleotide dependentprotein kinase, whereby growth of the axon is promoted; and detecting aresultant growth promotion of the axon.
 2. The method of claim 1,wherein the activator comprises an active component selected from acyclic nucleotide analog, an activator of a cyclic nucleotide cyclase, anitric oxide (NO) inducer and an inhibitor of a cyclic nucleotidephosphodiesterase.
 3. The method of claim 1, wherein the activatorcomprises an active component selected from: (a) an activator of acyclic nucleotide cyclase selected from an adenylate cyclase activatorselected from forskolin, 7β-deaceyl-7β-[γ-(morpholino)butyryl]-forskolinand 6β-[β′-(piperidino)-propionyl]-forskolin; and a guanylate cyclaseactivator which is protoporphyrin-9 (PP-9); (b) a cyclic nucleotideanalog selected from a protein kinase-A (PKA) activator selected from8-bromo-adenosine 3′,5′-monophosphate (8-Br-cAMP), 8-chloro-adenosine3′,5′-monophosphate (8-Cl-cAMP), 8-(4-chlorophenylthio)-cAMP,dibutyryl-cAMP, dioctanoyl-cAMP, Sp-cAMPS and Sp-8-bromo-cAMPS; and aprotein kinase G (PKG) activator selected from 8-br-cGMP,8-(4-chlorophenylthio)-cGMP and dibutyryl-cGMP; (c) a NO inducer whichis an NO donor selected from S-nitroso-N-acetylpenicillamine (SNAP),Glyco-SNAP-1, Glyco-SNAP-2, 2,2′-(hydroxynitrosohydrazono)bis-ethanamine(NOC-18) and (+/−)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide(NOR-3); and (d) an inhibitor of a cyclic nucleotide phosphodiesteraseselected from 3-isobutyl-1-methylxanthine (IBMX) and rolipram.
 4. Themethod of claim 1, wherein the activator comprises an active componentthat is forskolin.
 5. The method of claim 1, wherein the activatorcomprises an active component that is7β-deaceyl-7β-[γ-(morpholino)butyryl]-forskolin.
 6. The method of claim1, wherein the activator comprises an active component that is6β-[β′-(piperidino)-propionyl]-forskolin.
 7. The method of claim 1,wherein the activator comprises an active component that isprotoporphyrin-9 (PP-9).
 8. The method of claim 1, wherein the activatorcomprises an active component that is 8-bromo-adenosine3′,5′-monophosphate (8-Br-cAMP).
 9. The method of claim 1, wherein theactivator comprises an active component that is 8-chloro-adenosine3′,5′-monophosphate (8-Cl-cAMP).
 10. The method of claim 1, wherein theactivator comprises an active component that is8-(4-chlorophenylthio)-cAMP.
 11. The method of claim 1, wherein theactivator comprises an active component that is dibutyryl-cAMP.
 12. Themethod of claim 1, wherein the activator comprises an active componentthat is dioctanoyl-cAMP.
 13. The method of claim 1, wherein theactivator comprises an active component that is Sp-cAMPS.
 14. The methodof claim 1, wherein the activator comprises an active component that isSp-8-bromo-cAMPS.
 15. The method of claim 1, wherein the activatorcomprises an active component that is 8-br-cGMP.
 16. The method of claim1, wherein the activator comprises an active component that is8-(4-chlorophenylthio)-cGMP.
 17. The method of claim 1, wherein theactivator comprises an active component that is dibutyryl-cGMP.
 18. Themethod of claim 1, wherein the activator comprises an active componentthat is S-nitroso-N-acetylpenicillamine (SNAP).
 19. The method of claim1, wherein the activator comprises an active component that isGlyco-SNAP-1.
 20. The method of claim 1, wherein the activator comprisesan active component that is Glyco-SNAP-2.
 21. The method of claim 1,wherein the activator comprises an active component that is2,2′-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18).
 22. The method ofclaim 1, wherein the activator comprises an active component that is(+/−)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3). 23.The method of claim 1, wherein the activator comprises an activecomponent that is 3-isobutyl-1-methylxanthine (IBMX). 24 The method ofclaim 1, wherein the activator comprises an active component that isrolipram.
 25. The method of claim 1, wherein the repulsion factorcomprises an active component selected from a semaphorin, a netrin, aMAG and a CNS myelin fraction.
 26. The method of claim 1, wherein theprotein kinase is protein kinase A or G.
 27. The method of claim 1,wherein the neuron is a corticospinal tract neuron.